2015 In the last year, our work has covered several major areas: I. Tec Kinases: Mutations affecting the Tec kinase, Btk, cause the genetic disorder X-linked Agammmaglobulimemia, characterized by abnormal B cell development and function. Over the last 15 years, we showed that the Tec kinases expressed in T lymphocytes, Itk and Rlk, are important modulators of T cell signaling: mutations of Itk and Rlk do not prevent T cell development and function, but alter outcomes by affecting T cell receptor signaling strength via activation of PLC-g and Ca++ mobilization. Confirming its importance, mutations of Itk have been described in a profound EBV-induced lethal immunodeficiency. Recently, we have focused on the effects of mutations on patterns of cytokine production by CD4+ T helper cells. In the last year, we focused on the role of Itk in the development of Th9 cells, important contributors to asthma and airway hypersensitivity. Previous work had demonstrated that Itk was required for Type II immune responses that lead to asthma. We have now shown that Itk is strictly required for the expression of IL-9 and link this defect to TCR signaling defects. We further show that IL-2 can rescue multiple defects in Itk-deficient T cells, including the induction of IRF4, a transcription factor linked to TCR signaling strength. Our work provides insight into mechanisms by which IL-2 promotes the function of sub-optimally activated T cells and suggests Itk as a therapeutic target for IL-9-mediated diseases (Gomez-Rodriguez et al, 2016). Furthermore, as an expert in T helper differentiation and in gene-targeting, Dr. Gomez-Rodriguez has contributed to the work of several other laboratories. II. Phosphoinositide 3 Kinase (PI3K) delta: As part of a collaborative study, we previously helped describe and characterize activating mutations affecting PI3Kdelta, a hematopoietic-specific member of the PI3K catalytic subunit in patients with sino-pulmonary infections, mucosal lymphoid nodules, decreased circulating lymphocytes, lymphoproliferation, and EBV viremia. Our work focused on characterization of CD8+ cell defects in these patients, which showed elevated activation of downstream PI3K targets, including increased pAKT, and mTOR downstream targets (Lucas et al, Nature Immunol. 2014). To further understand these defects, we have generated a mouse model and are using these mice to provide new insight into the requirements for PI3K in immune homeostasis and function. III. SAP and the regulation of Tfh cells and humoral immune responses: Another major focus of our work is SAP, mutations of which cause the genetic disorder X-linked proliferative syndrome (XLP1),characterized by fatal EBV-infection, lymphomas, and antibody defects. SAP is a small SH2 containing adaptor that binds phosphorylated tyrosine residues in the intracellular tails of SLAM family co-stimulatory receptors. We previously generated SAP-deficient mice, which recapitulate features of XLP, including increased T cell activation and decreased antibody production upon infection (Czar et al PNAS). Notably, SAP-/- T cells failed to provide essential signals for B cells to generate germinal centers and long-term antibody responses, the hallmarks of successful vaccination. This work has helped define the importance of a subset of T cells, Tfh cells, that are required to provide signals for B cells to form germinal centers (Qi et al, Nature, 2008; Cannons et al, Immunity, 2010). Our work has provided insight into the requirement for T:B cell interactions in the development and function of Tfh cells, the critical helper T cell population required for providing signals to B cells for germinal center formation and long-term humoral immunity, a key feature of protective responses to most immunizations. In the last year, we have used RNAseq to evaluation Tfh-specific gene-expression signatures. We found that the transcription factor TCF1, a component of the Wnt signaling pathway, is selectively expressed in Tfh cells in response to viral infection. Using conditional knockout mice and shRNA knockouts, we and others recently provided evidence that TCF1 is required for Tfh responses to viral infection (Wu et al, Cell Reports, 2015). Our work helps provide insight into the regulation of this important T helper cell population, which permits an organism to respond appropriately to distinct infectious organisms and vaccines (Cannons et al Trends Immunol. 2013). In recent work, we have found that similar signaling and transcriptional networks are required for longterm CD8 cell responses to chronic infection (Wu et al, submitted). IV. To increase our ability to probe the immune system, we have developed new CRISPR mediated tools to inactivate multiple genes in mice and in primary T cells (Huang et al PLOS One 2016). We are using these tools to probe function of the SLAM family members and of other genes involved in Tfh cell differentiation.